The physiologic aggresome mediates cellular inactivation of iNOS

Abstract

Nitric Oxide (NO), produced by inducible nitric oxide synthase (iNOS), has been implicated in the pathogenesis of various biological and inflammatory disorders. Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed ''physiologic aggresome,'' is exemplified by aggresome formation of iNOS, an important host defense protein. The functional significance of cellular formation of the iNOS aggresome is hitherto unknown. In this study, we used live cell imaging, fluorescence microscopy, and intracellular fluorescence NO probes to map the subcellular location of iNOS and NO under various conditions. We found that NO production colocalized with cytosolic iNOS but aggresomes containing iNOS were distinctly devoid of NO production. Further, cells expressing iNOS aggresomes produced significantly less NO as compared with cells not expressing aggresomes. Importantly, primary normal human bronchial epithelial cells, stimulated by cytokines to express iNOS, progressively sequestered iNOS to the aggresome, a process that correlated with marked reduction of NO production. These results suggest that bronchial epithelial cells used the physiologic aggresome mechanism for iNOS inactivation. Our studies reveal a novel cellular strategy to terminate NO production via formation of the iNOS aggresome.misfolded ͉ nitric oxide ͉ primary cells ͉ proteasome ͉ degradation

Transient Aggregation of Ubiquitinated Proteins Is a Cytosolic Unfolded Protein Response to Inflammation and Endoplasmic Reticulum Stress

“…In the cytosol, responses to unfolded proteins include chaperon refolding, aggresome sequestration, and protein degradation (2,5,6). In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated.…”

Phosphorylated TP63 Induces Transcription of RPN13, Leading to NOS2 Protein Degradation

“…We previously showed that RPN13 physically interacts with NOS2, modulating its activity through blocking its homodimerization and potentially targeting nascent NOS2 polypeptides into a membrane-bound location (21)(22)(23). NOS2 inactivation is also likely to be achieved by targeting this enzyme into a proteasome-dependent degradation pathway, modulating NOS2 turnover and protecting cells against longterm and high-output NO production by this powerful enzyme (5)(6)(7)(8).…”

“…Cisplatin Induces the Phospho-⌬Np63␣/RPN13-dependent Proteasome Degradation of NOS2-RPN13 was previously found to bind NOS2 and to inactivate its function through 1,3,5,7,9,11,13, and 15) and 10 g/ml cisplatin (CIS; ϩ; samples 2,4,6,8,10,12,14, and 16) for 16 h were transfected with the pGL3 constructs for the 1750-bp RPN13 promoter and 5Ј-deletions. Samples 1 and 2, control (empty vector); samples 3 and 4, Ϫ1750/ϩ50 bp; samples 5 and 6, Ϫ1280/ϩ50 bp; samples 7 and 8, Ϫ1192/ϩ50 bp; samples 9 and 10, Ϫ1043/ϩ50 bp; samples 11 and 12, Ϫ901/ϩ50 bp; samples 13 and 14, Ϫ608/ϩ50 bp; samples 15 and 16, Ϫ331/ϩ50 bp.…”

“…There is emerging evidence that inhibiting the aggresome pathway leads to accumulation of misfolded proteins and to cell death (apoptosis or autophagy) in tumor cells (1)(2)(3)(4). One of these misfolded proteins, inducible NOS2, was shown to be targeted into an aggresome/proteasome pathway, which leads to termination of NO production by NOS2 (5)(6)(7)(8). NOS2 plays a critical role in massive NO production in a variety of cell types under physiologic and pathophysiologic conditions (9 -13).…”

Identification of a Flavin Mononucleotide Module Residue Critical for Activity of Inducible Nitrite Oxide Synthase

“…Regarding the general importance of this residue, we further studied the sensitivity of iNOS mutants to BBS-2, a selective iNOS dimerization inhibitor (25,26). We observed that 2 M BBS-2 decreased NO production around 75% by wild-type iNOS or iNOS mutants E658A, E658K, and E658Q (supplementary Fig.…”